ELISPOT assay to detect cytokine-secreting murine and human cells

Enzyme-linked immunospot assays (ELISPOT) were initially developed to detect and quantify individual antibody-secreting B cells. As the sensitivity of this assay improved, the much smaller amounts of cytokine and chemokine produced by individual T cells became amenable to detection. ELISPOT assays utilize high-affinity antibody pairs directed against different epitopes on a single cytokine/chemokine. The critical first step involves binding the highest affinity Ab to a solid matrix. The plates are blocked to prevent nonspecific interactions, and the cells of interest incubated in the Ab-coated wells, during which time they secrete the cytokine/chemokine of interest. The secreted protein binds to the Abs immediately below the producer cell. This bound protein is recognized by a secondary enzyme-linked Ab. A colorimetric substrate is used to generate a dark precipitate or "spot" that marks the position of the protein-producing cell. The resultant spots are permanent and can be quantified visually, microscopically, or electronically.